Throughout this application, various publications are referenced by author and year. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
The genetics of atherosclerosis has been the focus of intense investigation. A subset of cases is caused by uncommon Mendelian mutations that predispose individuals to atherosclerosis (Breslow 2000; Keating and Sanguinetti 1996; Lifton 1996). The mutated genes include low-density lipoprotein receptor (LDLR) (Hobbs et al. 1992), cystathionine beta-synthase (CBS) (Kraus 1999), and, in some cases, ATP-binding cassette-A1 (Bodzioch et al. 1999; Brooks-Wilson et al. 1999; Rust et al. 1999) among others. Identification of these genes has shed light on biochemical pathways involved in atherogenesis and provided the basis for current therapeutic interventions. However, the common forms of atherosclerosis are multifactorial in origin. Attempts to map the common susceptibility loci have been hampered by genetic heterogeneity, polygenic inheritance, incomplete pedigrees, and environmental influences. The fact that few of the genome-wide linkage studies have reported loci with large effects points to the existence of multiple loci each having small to moderate effects (Aouizerat et al. 1999; Hixson and Blangero 2000; Rice et al. 2000; Shearman 2000). The modest nature of susceptibility gene effects will likely require extremely large sample sizes or very densely-spaced genetic markers for successful linkage mapping (Risch and Merikangas 1996).
Mouse models offer significant advantages for genetic dissection of complex diseases. The ability to perform selective breeding, produce many offspring, determine inheritance of alleles without ambiguity, and control the environment is a critical factor. Early studies of murine atherosclerosis indicated that there was a clear genetic component. Inbred strains of mice exhibited a spectrum of aortic fatty streak lesion areas following the feeding of atherogenic diets high in cholesterol, fat, and cholic acid (Paigen et al. 1985; Qiao et al. 1994; Roberts and Thompson 1977). A number of susceptibility loci (Ath1-8) were reported based on phenotypic analyses of recombinant inbred strains derived from xe2x80x9cresistantxe2x80x9d and xe2x80x9csusceptiblexe2x80x9d parents (Paigen 1995; Paigen et al. 1987, 1989; Stewart-Phillips et al. 1989). Although these studies were instrumental in pointing out strain-specific variations, none of the loci have been confirmed by more rigorous analyses of large genetic crosses.
A shortcoming of the diet-fed, inbred mouse model (in terms of carrying out quantitative genetic studies) is that aortic lesion development is minimal even in susceptible strains. Recently, Dansky et al. (1999) showed that the strain-related differences in susceptibility could be accentuated when a gene-targeted disease model was employed. Thus, C57BL/6J mice homozygous for the apolipoprotein E knockout allele exhibited 7-9 fold greater aortic root lesion area relative to FVB/NJ mice homozygous for the allele without any overlap of the phenotypic values. To provide candidate susceptibility loci for human atherosclerosis, we have performed a genome scan of an interspecific cross using the low-density lipoprotein receptor knockout model (Ishibashi et al. 1993). In this model, feeding of a Western-style diet results in elevated plasma LDL levels (similar to levels in humans) and development of human-like complicated fibrous plaques (Masucci-Magoulas et al. 1997). Two significant susceptibility loci were localized to chromosome (Chr) 4 and 6. The effects of these loci were independent of common risk factors for human disease including plasma lipoprotein levels, plasma insulin levels, and body weight.
This invention provides an isolated nucleic acid encoding a mammalian LOX-1 receptor protein, wherein the receptor protein comprises consecutive amino acids having the following sequence: xe2x80x94S, X, X, E, L, K, X, X, I, X, T, X, X, X, K, L, X, E, K, S, K, E, Q, X, E, L, X, X, X, X, X, N, L, Q, E, X, L, X, R, X, A, N, X, Sxe2x80x94 (SEQ ID NO: 39), wherein X is any amino acid.
The invention provides an isolated nucleic acid encoding a mammalian membrane-bound LOX-1 receptor protein, wherein the nucleic acid encodes a protein selected from the group consisting of:
(a) a LOX-1 receptor protein comprising consecutive amino acids having a sequence identical to that set forth for Isoform 1 in SEQ ID NO: 20,
(b) a LOX-1 receptor protein comprising consecutive amino acids having a sequence identical to that set forth for Isoform 3 in SEQ ID NO: 24, and
(c) a LOX-1 receptor protein comprising consecutive amino acids having a sequence identical to that set forth for Isoform 4 in SEQ ID NO: 26.
The invention provides an isolated nucleic acid encoding a mammalian soluble LOX-1 receptor protein, wherein the nucleic acid encodes a protein selected from the group consisting of:
(a) a LOX-1 receptor protein comprising consecutive amino acids having a sequence identical to that set forth for Isoform 7 in SEQ ID NO: 14,
(b) a LOX-1 receptor protein comprising consecutive amino acids having a sequence identical to that set forth for Isoform 8 in SEQ ID NO: 16, and
(c) a LOX-1 receptor protein comprising consecutive amino acids having a sequence identical to that set forth for Isoform 9 in SEQ ID NO: 18.
The invention provides an isolated nucleic acid encoding a mammalian LOX-1 receptor protein, wherein the nucleic acid comprises:
(a) a nucleic acid sequence given in any one of SEQ ID Nos: 13, 15, 17, 19, 21, 23, 25, 27, or 28; or
(b) a nucleic acid sequence degenerate to a sequence of (a) as a result of the genetic code.
The invention provides a method involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LOX-1 receptor, which comprises contacting cells expressing on their cell surface the mammalian LOX-1 receptor with both the chemical compound and a second chemical compound known to bind to the receptor, and separately with only the second chemical compound, under conditions suitable for binding of both compounds, and measuring specific binding of the second chemical compound to the mammalian LOX-1 receptor, a decrease in the binding of the second chemical compound to the mammalian LOX-1 receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LOX-1 receptor.
The invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian LOX-1 receptor to identify a compound which specifically binds to the mammalian LOX-1 receptor, which comprises:
(a) contacting cells expressing on their cell surface the mammalian LOX-1 receptor with the plurality of compounds not known to bind specifically to the mammalian LOX-1 receptor, under conditions permitting binding of compounds known to bind to the mammalian LOX-1 receptor;
(b) determining whether the binding of a compound known to bind to the mammalian LOX-1 receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so
(c) separately determining the binding to the mammalian LOX-1 receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian LOX-1 receptor.
The invention provides a method of identifying a compound which activates a mammalian LOX-1 receptor which comprises contacting cells expressing on their cell surface the mammalian LOX-1 receptor with the compound under conditions permitting activation of the LOX-1 receptor, and detecting activation of the LOX-1 receptor, thereby identifying the compound as a compound which activates a mammalian LOX-1 receptor.
The invention provides a method of identifying a compound which inhibits the activity of a mammalian LOX-1 receptor which comprises contacting cells expressing on their cell surface the mammalian LOX-1 receptor with the compound under conditions permitting inhibition of the activity of the LOX-1 receptor, and detecting inhibition of the activity of the LOX-1 receptor, thereby identifying the compound as a compound which inhibits the activity of a mammalian LOX-1 receptor.
The invention provides a method of screening a plurality of chemical compounds not known to activate a mammalian LOX-1 receptor to identify a compound which activates the mammalian LOX-1 receptor which comprises:
(a) contacting cells expressing on their cell surface the mammalian LOX-1 receptor with the plurality of compounds not known to activate the mammalian LOX-1 receptor, under conditions permitting activation of the mammalian LOX-1 receptor;
(b) determining whether the activity of the mammalian LOX-1 receptor is increased in the presence of the compounds; and if so
(c) separately determining whether the activation of the mammalian LOX-1 receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the mammalian LOX-1 receptor.
The invention provides a method of screening a plurality of chemical compounds not known to inhibit the activity of a mammalian LOX-1 receptor to identify a compound which inhibits the activity of the mammalian LOX-1 receptor, which comprises:
(a) contacting cells expressing on their cell surface the mammalian LOX-1 receptor with the plurality of compounds in the presence of a known compound which activates the mammalian LOX-1 receptor, under conditions permitting activation of the mammalian LOX-1 receptor;
(b) determining whether the activity of the mammalian LOX-1 receptor is reduced in the presence of the plurality of compounds, relative to the activity of the mammalian LOX-1 receptor in the absence of the plurality of compounds; and if so
(c) separately determining the inhibition of activity of the mammalian LOX-1 receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activity of the mammalian LOX-1 receptor.
The invention provides a method of treating or preventing atherosclerosis in a subject which comprises administering to the subject an amount of a compound effective to decrease the activity of a mammalian LOX-1 receptor and treat atherosclerosis in the subject.
The invention provides a method of determining the susceptibility of a subject to atherosclerosis, which comprises detecting soluble LOX-1 receptor in the subject""s plasma, wherein the presence of soluble LOX-1 receptor indicates an decreased susceptibility to atherosclerosis and an absence of soluble LOX-1 receptor indicates an increased susceptibility to atherosclerosis.
The invention provides a method of treating inflamation in a subject which comprises administering to the subject an amount of a soluble mammalian LOX-1 receptor effective to treat inflamation in the subject.
The invention provides a method of treating inflammation in a subject which comprises administering to the subject an amount of a compound effective to decrease the activity of a mammalian LOX-1 receptor and treat inflammation in the subject.
The invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LOX-1 receptor, which comprises administering to the subject an amount of a compound effective to decrease the activity of the LOX-1 receptor, thereby treating the abnormality.
The invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing LOX-1 signal transduction, which comprises administering to the subject an amount of a soluble mammalian LOX-1 receptor effective to bind LOX-1 receptor ligand and reduce availability of LOX-1 receptor ligand to bind to a membrane-bound LOX-1 receptor, thereby decreasing LOX-1 signal transduction and treating the abnormality.